Dendritic cells (DCs) are powerful antigen-presenting cells with a unique ability to induce primary T cell responses. The availability of immature human DCs is critical for the study of antigen processing and presentation, and for understanding the mechanisms of inducing immunity and tolerance. Functional analysis of human DC subsets was significantly facilitated by development of an in vitro system for DC differentiation from CD34+ hematopoietic stem cells and monocytes. However, obtaining large numbers of human DC progenitors is still a laborious process and poses potential risks for donors. Human embryonic stem (hES) cells represent a unique population of cells capable of self-renewal and differentiation, and directed hematopoietic differentiation of hES cells reproduces many aspects of embryonic hematopoiesis. Therefore, hES cells can be used as a unique source of hematopoietic and DC precursors to address the essential questions of DC development. In addition, since ES cells can be expanded without limit, they can be seen as an alternative scalable source of cells for DC vaccines or DC-mediated induction of immune tolerance. We recently developed a method for the efficient differentiation of hES cells into hematopoietic cells and DCs utilizing hES cell/OP9 coculture. The overall goal of this proposal is to apply the described model to identify the population of myeloid DC precursors, which can be expanded through activation of the Wnt/[unreadable]-catenin pathway. In the first specific aim, we will characterize the hierarchy of hES cell-derived myeloid DC precursors. In the second specific aim we will evaluate the effect of [unreadable]-catenin on the proliferation and differentiation of hES cell-derived myeloid DC progenitors at different stages of maturation using a tamoxifen-inducible promoter system to conditionally express [unreadable]-catenin. Eventually, these studies will assist with developing protocols for the production of DCs from hES cells in large quantities and with desired functional characteristics. In addition, accomplishment of the above-listed aims will provide new insights into cellular pathways of DC development and on how the Wnt/[unreadable]-catenin pathway influences the survival and proliferation of hES cell-derived myeloid DC progenitors. [unreadable] [unreadable] [unreadable]